Lipofectamine LTX® Reagent performs well with vector-based RNAi experiments. For siRNA and Stealth RNAi transfections, we recommend Lipofectamine RNAiMAX. Go to www.lifetechnologies.com/RNAi or contact Technical Service for more information.

Transfecting HEK 293 Cells

Use this procedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, below). All amounts and volumes are given on a per well basis.

The day before transfection, trypsinize and count the cells. Plate 0.5 -1.25x105 cells per well in 0.5 ml of complete growth medium. Cell density should be 50-80% confluent on the day of transfection.

(Optional) The day of transfection, remove growth medium from cells and replace with 0.5 ml of complete growth medium.

For each well of cells to be transfected, dilute 0.5 μg of DNA in 100 μl of Opti-MEM® I Reduced Serum Media without serum.

For each well of cells, add 0.75-1.75 μl of Lipofectamine LTX® Reagent into the above diluted Opti-MEM®:DNA solution, mix gently and incubate 30 minutes at room temperature to form DNA- Lipofectamine LTX® Reagent complexes.

After 30 minute incubation, add 100 μl of the DNA- Lipofectamine LTX® Reagent complexes directly to each well containing cells and mix gently by rocking the plate back and forth.

Complexes do not have to be removed following transfection. Incubate the cells at 37oC in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression.